US8697382B2 - Method of identifying metallo-β-lactamase-producing bacteria - Google Patents

Method of identifying metallo-β-lactamase-producing bacteria Download PDF

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US8697382B2
US8697382B2 US13/260,965 US201013260965A US8697382B2 US 8697382 B2 US8697382 B2 US 8697382B2 US 201013260965 A US201013260965 A US 201013260965A US 8697382 B2 US8697382 B2 US 8697382B2
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metallo
lactamase
alkyl group
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Akihiro Morinaka
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Meiji Seika Pharma Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)

Definitions

  • the present invention relates to a method of identifying metallo- ⁇ -lactamase-producing bacteria, particularly, a method of identifying a type of metallo- ⁇ -lactamase-producing bacteria.
  • the present invention also relates to a kit for the identification of metallo- ⁇ -lactamase-producing bacteria, and an identification method using the kit.
  • Antimicrobial drugs such as carbapenem, new quinolone, and aminoglycoside are used for serious infections caused by opportunistic pathogens such as Pseudomonas aeruginosa .
  • bacteria that have also acquired resistance to these drugs have been reported in recent years.
  • Pseudomonas aeruginosa that has acquired resistance to these 3 drugs is called multidrug resistance Pseudomonas aeruginosa (MDRP) and has particularly been viewed as a problem in clinical practice. It has been reported that once MDRP is separated in clinical practice, the same strain spreads in the same sickbed or hospital.
  • MBL Metallo- ⁇ -lactamase
  • MBL multidrug resistance bacteria
  • Enterobacteriaceae carrying this plasmid transmits the plasmid to Pseudomonas aeruginosa , which is in turn rendered resistant to ⁇ -lactam agents.
  • MDRP carrying this plasmid transmits the plasmid to Enterobacteriaceae, possibly resulting in the emergence of new MBL-producing enterobacteria.
  • any of MBL-producing bacteria have the risk of causing the emergence of new multidrug resistance bacteria.
  • MBL-producing bacteria is considered as an important issue in medical practice.
  • the identification of responsible bacteria is inevitable for the treatment of infections.
  • the responsible bacteria are multidrug resistance bacteria, the early selection of effective drugs and coping plans is required.
  • early treatment can be achieved in an appropriate manner by conveniently detecting MBL.
  • these plasmid-mediated MBLs mainly include two types: IMP and VIM types. Depending on the enzyme type, some drugs exhibit sensitivity. Thus, a drug suitable for treatment may be selected rapidly by identifying an IMP or VIM type.
  • MBL inhibitor-containing antimicrobial drugs currently under study or ⁇ -lactam agents stable for MBL are considered to differ in inhibitory activity or effectiveness between IMP and VIM MBL-producing bacteria. The identification of the enzyme type is very useful for selection criteria for these drugs developed in the future.
  • a method of identifying an IMP or VIM type can be used worldwide.
  • IMP or VIM MBL-producing bacteria can be identified without using specialized machines such as PCR machines, such identification seems to be also useful for various epidemiologic studies or for the preparation of antibiograms in individual facilities in preparation for outbreaks.
  • Japanese Patent No. 3754993 discloses a method of determining whether or not bacteria to be detected are MBL-producing bacteria.
  • Japanese Patent Laid-Open Publication No. 2001-299388 discloses a method of testing the drug sensitivity of MBL-producing bacteria by a broth microdilution method using the combination of a liquid medium containing a ⁇ -lactam agent and a liquid medium containing a ⁇ -lactam agentimetallo- ⁇ -lactamase inhibitor.
  • 2004-166694 (SHOWA YAKUHIN KAKO CO., LTD.) discloses a detection method of rapidly identifying ⁇ -lactamase and a kit therefor.
  • these documents neither disclose nor suggest a method of identifying a type of MBL-producing bacteria or a method of simultaneously performing the detection of MBL-producing bacteria and the identification of a type of the MBL-producing bacteria.
  • a metallo- ⁇ -lactamase inhibitor represented by the formula (I) used in the present invention is a compound disclosed in Japanese Patent No. 4122049. It is known that the compound represented by the formula (I) in combined use with a ⁇ -lactam antibiotic strengthens effectiveness for metallo- ⁇ -lactamase-producing resistance bacteria in the treatment of bacterial infection.
  • metallo- ⁇ -lactamase-producing bacteria can be detected by spotting a metallo- ⁇ -lactamase inhibitor represented by the formula (I) onto a surface of a solid medium coated with the bacteria to be tested, spotting 3 types of ⁇ -lactam agents at 3 respective positions different from the spot of the metallo- ⁇ -lactamase inhibitor, culturing the solid medium, and then determining the shape of an inhibition zone formed around the spot of each of the ⁇ -lactam agents; and an IMP or VIM type of the metallo- ⁇ -lactamase-producing bacteria can also be identified by these procedures.
  • the present invention is based on these findings.
  • An object of the present invention is to provide a method of identifying metallo- ⁇ -lactamase-producing bacteria, particularly, a method of conveniently identifying a type of metallo- ⁇ -lactamase-producing bacteria.
  • Another object of the present invention is to provide a kit for use in the identification of metallo- ⁇ -lactamase-producing bacteria and an identification method using the kit.
  • a method of identifying metallo- ⁇ -lactamase-producing bacteria comprising spotting metallo- ⁇ -lactamase inhibitor represented by the following formula (I) onto a surface of a solid medium coated with the bacteria to be tested, further spotting at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase, at positions different from the spot of the metallo- ⁇ -lactamase inhibitor, culturing the solid medium, and then identifying the metallo- ⁇ -lactamase-producing bacteria based on the shape of an inhibition zone formed around the spot of each of the ⁇ -lactam agents:
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and
  • two M 1 which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • a method of identifying a type of metallo- ⁇ -lactamase-producing bacteria to be tested comprising spotting a metallo- ⁇ -lactamase inhibitor represented by the following formula (I) onto a surface of a solid medium coated with the bacteria to be tested, further spotting at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase, at positions different from the spot of the metallo- ⁇ -lactamase inhibitor, culturing the solid medium, and then identifying the type of the metallo- ⁇ -lactamase-producing bacteria to be tested based on the shape of an inhibition zone formed around the spot of each of the ⁇ -lactam agents:
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and
  • two M 1 which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • a kit for use in the identification of metallo- ⁇ -lactamase-producing bacteria comprising: a substrate having three strips extended in different directions; a disk containing a metallo- ⁇ -lactamase inhibitor represented by the following formula (I), the disk being disposed at the center of the substrate; and three disks respectively containing (A) a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, (B) a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase, and (C) a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, the three disks being disposed on the three respective strips of the substrate
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and
  • two M 1 which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • a method of identifying metallo- ⁇ -lactamase-producing bacteria comprising placing the kit for use in the identification of metallo- ⁇ -lactamase-producing bacteria according to the present invention onto a surface of a solid medium coated with the bacteria to be tested, culturing the bacteria, and then identifying the metallo- ⁇ -lactamase-producing bacteria based on the shape of an inhibition zone formed around each of the disks of the ⁇ -lactam agents.
  • the present invention also relates to the following (1) to (9):
  • a method of detecting metallo- ⁇ -lactamase-producing bacteria comprising spotting metallo- ⁇ -lactamase inhibitor represented by the following compound (I) onto a surface of a solid medium coated with the bacteria to be tested, further spotting (A) a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, (B) a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase, and (C) a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, at 3 respective positions different from the spot of the following compound (I), culturing the solid medium, and then identifying the metallo- ⁇ -lactamase-producing bacteria
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and two M 1 , which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • a method of identifying an IMP or VIM type of metallo- ⁇ -lactamase-producing bacteria to be tested comprising spotting a metallo- ⁇ -lactamase inhibitor represented by the following compound (I) onto a surface of a solid medium coated with the bacteria to be tested, further spotting (A) a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, (B) a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase, and (C) a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, at 3 respective positions different from the spot of the following compound (I), culturing the solid medium, and then identifying the
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and two M 1 , which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • a kit for use in the detection of metallo- ⁇ -lactamase-producing bacteria comprising: a substrate having three strips extended in different directions; a disk containing a metallo- ⁇ -lactamase inhibitor represented by the compound (I), the disk being disposed at the center of the substrate; and three disks respectively containing (A) a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, (B) a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase, and (C) a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase, the three disks being disposed on the three respective strips of the substrate at an equal distance from the disk of the
  • a method of detecting metallo- ⁇ -lactamase-producing bacteria comprising placing a kit according to (8) onto a surface of a solid medium coated with the bacteria to be tested, culturing the bacteria, and then identifying the metallo- ⁇ -lactamase-producing bacteria based on the minimum and maximum values of the inhibition zone formed around each of the disks of the ⁇ -lactam agents (A) to (C) and the degree of change in radius determined from both the values.
  • MBL plasmid-mediated enzyme
  • IMP plasmid-mediated enzyme
  • VIM plasmid-mediated enzyme
  • MBL-producing bacteria can be detected by a convenient method without using specialized machines such as PCR machines. Furthermore, either an IMP or VIM type of the detected MBL-producing bacteria can also be identified. Thus, drugs suitable for treatment can be selected rapidly. By extension, the administration of ineffective drugs can be prevented, probably leading to reduction in physical or mental burdens to patients and in medical expenses.
  • MBL inhibitor-containing antimicrobial drugs currently under study or ⁇ -lactam agents stable for MBL differ in inhibitory activity or effectiveness between IMP and VIM MBL-producing bacteria in some cases.
  • the identification method of the present invention is very useful for selection criteria for these drugs developed in the future.
  • the identification method of the present invention is also very useful for various epidemiologic studies or for the preparation of antibiograms in individual facilities in preparation for outbreaks.
  • FIG. 1 is a diagram showing measured positions in an inhibition zone.
  • FIG. 2 is a flowchart showing the identification of an MBL type.
  • FIG. 3 is a diagram illustrating a kit of the present invention.
  • FIG. 4 is a photograph of an inhibition zone on a solid medium obtained in Example 2.
  • FIG. 5 is a photograph of an inhibition zone on a solid medium obtained in Example 3.
  • FIG. 6 shows the results of identification of the MBL type of Example 1 using the flowchart in FIG. 2 .
  • whether or not bacteria to be tested are MBL-producing bacteria, and, further, an IMP or VIM type or any other type of the identified MBL-producing bacteria can be determined based on the shape of an inhibition zone formed on a solid medium.
  • the method according to the present invention can be carried out by coating a surface of a solid medium with bacteria to be tested, then spotting thereonto a metallo- ⁇ -lactamase inhibitor represented by the formula (I), further spotting at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase, at respective positions different from the spot of the metallo- ⁇ -lactamase inhibitor, and culturing the solid medium.
  • a solid medium that adheres to the standard method specified by the Japanese Society of Chemotherapy or the disk diffusion method specified by CLSI can be used in the present invention.
  • Examples thereof include Muller-Hinton agar media (BD Difco).
  • the form of the solid medium is not particularly limited as long as the method according to the present invention can be carried out.
  • a solid medium of approximately 4 mm in thickness can be used.
  • the coating of a surface of a solid medium with bacteria to be tested can be performed by a method that adheres to the standard method specified by the Japanese Society of Chemotherapy or the disk diffusion method specified by CLSI.
  • the coating with the bacteria can be performed by uniformly smearing a bacterial solution adjusted to McFarland standard turbidity of 0.5 over the surface of the solid medium using a sterile swab or the like.
  • a metallo- ⁇ -lactamase inhibitor represented by the formula (I) and at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase can be spotted onto the surface of the solid medium coated with the bacteria to be tested.
  • R 1 represents a C 2-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent;
  • R 2 represents a C 1-6 alkyl group, a C 3-7 cyclic alkyl group, a hydroxymethyl group, a —C 1-3 alkylene-phenyl group, a —C 0-1 alkylene-heterocyclic ring, a —O—C 1-6 alkyl group, or a —S—C 1-6 alkyl group, and all of these groups may have a substituent; and
  • two M 1 which may be the same or different, represent a hydrogen atom, a pharmaceutically acceptable cation, or a pharmaceutically acceptable group that can be hydrolyzed in vivo.
  • C 1-6 ”, “C 2-6 ”, “C 3-7 ”, “C 1-3 ”, and “C 0-1 ” represent the number of carbon atoms.
  • a “C 1-6 alkyl group” refers to an alkyl group having 1 to 6 carbon atoms.
  • C 0 refers to a bond.
  • the term “lower” preferably refers to C 1-6 .
  • halogen atom refers to a fluorine, chlorine, bromine, or iodine atom.
  • heteroatom refers to a nitrogen, oxygen, or sulfur atom.
  • alkyl group or “alkoxy group” as a group or a moiety of a group means a linear or branched alkyl or alkoxy group.
  • a lower alkyl group preferably refers to a C 1-6 alkyl group, which is as defined above.
  • cyclic alkyl group as a group or a moiety of a group means a monocyclic alkyl group.
  • a lower cycloalkyl group preferably refers to a C 3-7 cyclic alkyl group, which is as defined above.
  • heterocyclic ring examples include 5- to 14-membered monocyclic to tricyclic heterocyclic rings containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur atoms, more preferably 5- to 10-membered monocyclic or bicyclic heterocyclic rings containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur atoms.
  • Preferable specific examples thereof include tetrahydrofuran, furan, pyrrolidine, piperidine, pyrazolidine, imidazolidine, piperazine, morpholine, thiomorpholine, pyrrole, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazole, tetrazole, thiadiazole, azetidine, thiazoline, quinuclidine, triazine, isobenzofuran, indole, indolizine, chromene, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, phthalazine, purine, and pteridine.
  • aryl preferably refers to an aromatic ring such as phenyl or substituted phenyl and a condensed ring such as naphthyl, phenanthrenyl, fluorenyl, or anthryl.
  • the aryl group is preferably phenyl, naphthyl, and fluorenyl groups.
  • the phrase “may have a substituent” means that the group may have preferably 1 to 6, more preferably 1 to 3 substituents.
  • the “substituent” is a hydroxy group, a thiol group, a, C 1-6 alkyl group, a C 1-6 alkoxy group, an S—C 1-6 alkyl group, an amino group, a mono-substituted amino group, a di-substituted amino group, an amide group, a guanidyl group, an N-substituted amide group, an N,N-di-substituted amide group, a halogen atom, a carboxyl group, a phenyl group, a substituted phenyl group, a C 1-6 alkylcarbonyl group, a heterocyclic ring, a heterocyclic carbonyl group, or the like.
  • the phenyl may be condensed with another ring.
  • the substituent include a hydroxy group, a C 1-6 alkyl group, a C 1-6 alkoxy group, an amino group, a mono-substituted amino group, a di-substituted amino group, an amide group, a guanidyl group, an N-substituted amide group, an N,N-di-substituted amide group, a halogen atom, a carboxyl group, a phenyl group (the phenyl may be condensed with another ring), a substituted phenyl group, a C 1-6 alkylcarbonyl group, a heterocyclic ring, and a heterocyclic carbonyl group.
  • alkyl and alkoxy groups as a moiety of a group in the “substituent” such as the C 1-6 alkyl, C 1-6 alkoxy, S—C 1-6 alkyl, and C 1-6 alkylcarbonyl groups are as defined above.
  • the halogen atom is also as defined above.
  • the C 1-6 alkyl and C 1-6 alkoxy groups may be substituted by a hydroxy group, an amino group, a mono-substituted amino group, a di-substituted amino group, an amide group, a guanidyl group, an N-substituted amide group, an N,N-di-substituted amide group, a carboxyl group, a heterocyclic ring, a phenyl group, a substituted phenyl group, or the like.
  • this carboxyl group may be a pharmaceutically acceptable cation or a pharmaceutically acceptable group that can be hydrolyzed in vivo, and is preferably, for example, sodium salt or potassium salt.
  • substituted in the “substituent” such as the mono-substituted amino, di-substituted amino, amide, N-substituted amide, N,N-di-substituted amide, and substituted phenyl groups means that the group preferably has the “substituent” exemplified above.
  • heterocyclic ring in the heterocyclic ring and the heterocyclic carbonyl group as the “substituent” is as defined in the “heterocyclic ring” exemplified above.
  • heterocyclic carbonyl examples include morpholylcarbonyl, piperazylcarbonyl, and piperidylcarbonyl, preferably morpholyl-4-yl-carbonyl, piperazin-4-ylcarbonyl, and (4-hydroxypiperazin)-1-ylcarbonyl.
  • the “C 2-6 alkyl group” represented by R 1 may be linear or branched. Examples thereof include ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, neopentyl, i-pentyl, t-pentyl, n-hexyl, and i-hexyl groups.
  • the C 2-6 alkyl group is preferably a C 2-4 alkyl group, more preferably an ethyl group. This alkyl group may be substituted. Examples of the substituent include the “substituent” exemplified above, more preferably a hydroxy group, a thiol group, an amino group, and a halogen atom.
  • the “C 1-6 alkyl group” represented by R 2 may be linear or branched. Examples thereof include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, neopentyl, i-pentyl, t-pentyl, n-hexyl, and i-hexyl groups.
  • the C 1-6 alkyl group is preferably a C 2-4 alkyl group, more preferably an ethyl group. This alkyl group may be substituted. Examples of the substituent include the “substituent” exemplified above, more preferably a thiol group, an amino group, and a halogen atom.
  • C 3-7 cyclic alkyl group represented by R 1 or R 2 include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl groups, more preferably cyclopentyl, cyclohexyl, and cycloheptyl groups.
  • This C 3-7 cyclic alkyl group may be substituted.
  • substituent include the “substituent” exemplified above, more preferably a hydroxy group, a thiol group, a C 1-6 alkyl group, an amino group, and a halogen atom.
  • this C 3-7 cyclic alkyl group may be condensed with another ring, for example, aryl, preferably phenyl.
  • Examples of the “—C 1-3 alkylene-phenyl group” represented by R 1 or R 2 include benzyl, phenethyl, and phenylpropyl groups.
  • the phenyl group in the —C 1-3 alkylene-phenyl group may be substituted.
  • substituents examples include the “substituent” exemplified above, more preferably a hydroxy group, a C 1-6 alkyl group, —COOM (wherein M represents a hydrogen atom or a pharmaceutically acceptable cation), —CO—NR 22 R 23 (wherein R 22 and R 23 , which may be the same or different, represent a hydrogen atom or a C 1-6 alkyl group (preferably a C 1-4 alkyl group, more preferably a C 1-2 alkyl group) (this alkyl group may further be substituted by an aminocarbonyl group) or R 22 and R 23 , together with the nitrogen atom bonded thereto, may represent a 5- or 6-membered saturated heterocyclic ring comprising 1 to 2 oxygen or nitrogen atoms (which preferably refers to a morphonyl group, a piperazyl group, or a piperidyl group) (this heterocyclic ring, particularly, the piperidyl group, may be substituted by a hydroxy
  • the “—C 0-1 alkylene-heterocyclic ring” represented by R 1 or R 2 refers to a -bond-heterocyclic ring or a -methylene-heterocyclic ring.
  • the “heterocyclic ring” is as defined above, and preferable examples thereof include 5- to 10-membered monocyclic or bicyclic heterocyclic rings containing 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur atoms, more preferably 5- to 6-membered saturated or unsaturated heterocyclic rings comprising one nitrogen or oxygen atom.
  • heterocyclic ring examples include tetrahydrofuran, furan, pyrrolidine, piperidine, pyrazolidine, imidazolidine, piperazine, morpholine, thiomorpholine, pyrrole, thiophene, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole, pyridine, pyridazine, pyrimidine, pyrazine, triazole, tetrazole, thiadiazole, azetidine, thiazoline, quinuclidine, triazine, isobenzofuran, indole, indolizine, chromene, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, phthalazine, purine, and pteridine.
  • the bond or the methylene group may be bonded to any position on the heterocyclic ring.
  • One or more hydrogen atoms on this heterocyclic ring on the —C 0-1 alkylene-heterocyclic ring may be substituted.
  • the substituent include the “substituent” exemplified above, more preferably a hydroxy group, a thiol group, a C 1-6 alkyl group, an amino group, and a halogen atom.
  • the “—O—C 1-6 alkyl group” represented by R 1 or R 2 is a C 1-6 alkoxy group which may be linear, branched, or cyclic, preferably a —O—C 1-4 alkyl group. Examples thereof include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, s-butoxy, and t-butoxy, more preferably methoxy, ethoxy, propoxy, isopropoxy, and t-butoxy.
  • the alkyl moiety in this group may be substituted.
  • substituents examples include the “substituent” exemplified above, more preferably a hydroxy group, a thiol group, a C 1-6 alkyl group, an amino group, a halogen atom, and a phenyl group.
  • the “—S—C 1-6 alkyl group” represented by R 1 or R 2 is a C 1-6 alkylthio group which may be linear, branched, or cyclic, preferably a —S—C 1-4 alkyl group. Examples thereof include methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, s-butylthio, and t-butylthio, more preferably methylthio, ethylthio, propylthio, isopropylthio, and t-butylthio.
  • the alkyl moiety in this group may be substituted.
  • substituents examples include the “substituent” exemplified above, more preferably a hydroxy group, a thiol group, a C 1-6 alkyl group, an amino group, a halogen atom, and a phenyl group.
  • the “pharmaceutically acceptable cation” represented by M 1 is a cation that is capable of forming a salt with either or both of the carboxyl groups in the formula (I).
  • examples thereof include alkali metals, alkaline-earth metals, ammonium, and organic bases, preferably lithium, sodium, potassium, magnesium, calcium, ammonium, ethanolamine, triethanolamine, trimethylamine, triethylamine, and diisopropylamine.
  • the “pharmaceutically acceptable group that can be hydrolyzed in vivo” represented by M 1 refers to an eliminable group that is bonded to either or both of the carboxyl groups in the formula (I) and also refers to a group that is hydrolyzed and eliminated in vivo through metabolism to form a carboxyl group.
  • the “pharmaceutically acceptable group that can be hydrolyzed in vivo” is preferably an ester residue.
  • Examples thereof include those routinely used, such as a lower alkyl group, a lower alkenyl group, a lower alkylcarbonyloxy-lower alkyl group, a lower cycloalkylcarbonyloxy-lower alkyl group, a lower cycloalkylmethylcarbonyloxy-lower alkyl group, a lower alkenylcarbonyloxy-lower alkyl group, an arylcarbonyloxy-lower alkyl group, a tetrahydrofuranylcarbonyloxymethyl group, a lower alkoxy-lower alkyl group, a lower alkoxy-lower alkoxy-lower alkyl group, an arylmethyloxy-lower alkyl group, an arylmethyloxy-lower alkoxy-lower alkyl group, a lower alkoxycarbonyloxy-lower alkyl group, a lower alkoxycarbonyloxy-lower alkoxy group, a lower alkoxycarbonyl
  • the “pharmaceutically acceptable group that can be hydrolyzed in vivo” is preferably, for example, methyl, ethyl, 1-(cyclohexyloxycarbonyloxy)ethyl, acetoxymethyl, 1-(isopropyloxycarbonyloxy)ethyl, 1-(ethoxycarbonyloxy)ethyl, pivaloyloxymethyl, cyclohexyloxycarbonyloxymethyl, 1-(isobutyloxycarbonyloxy)ethyl, 1-(cyclohexyloxycarbonyloxy)-2-methylpropan-1-yl, isobutyloxycarbonyloxymethyl, isopropyloxycarbonyloxymethyl, isobutyryloxymethyl, (pentan-1-yl)oxycarbonyloxymethyl, (butan-1-yl)oxycarbonyloxymethyl, (1-ethyl propan-1-yl)oxycarbonyloxymethyl, isopentyloxycarbonyloxymethyl, (
  • R 1 is preferably a C 2-6 alkyl group or a C 3-7 cyclic alkyl group, more preferably a C 2-4 alkyl group, even more preferably an ethyl group.
  • R 2 is preferably a C 1-6 alkyl group or a C 3-7 cyclic alkyl group, more preferably a C 2-4 alkyl group, even more preferably an ethyl group.
  • two M 1 which may be the same or different, are preferably a hydrogen atom or a pharmaceutically acceptable cation, more preferably a sodium cation or a potassium cation.
  • the metallo- ⁇ -lactamase inhibitor used in the present invention is a compound of the formula (I) wherein R 1 is a C 2-6 alkyl group or a C 3-7 cyclic alkyl group; R 2 is a C 1-6 alkyl group or a C 3-7 cyclic alkyl group; and two M 1 , which may be the same or different, are a hydrogen atom or a pharmaceutically acceptable cation.
  • the metallo- ⁇ -lactamase inhibitor used in the present invention is a compound of the formula (I) wherein R 1 is a C 2-4 alkyl group; R 2 is a C 2-4 alkyl group; and two M 1 are a sodium cation or a potassium cation.
  • the metallo- ⁇ -lactamase inhibitor used in the present invention is a compound of the formula (I) wherein R 1 is an ethyl group; R 2 is an ethyl group; and two M 1 are a sodium cation or a potassium cation.
  • the compound of the formula (I) can be produced according to the description of Japanese Patent No. 41222049.
  • Examples of the “at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase” used in the present invention include: a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase; a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase; a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase; and a ⁇ -lactam agent that is easily influenced by VIM metallo- ⁇ -lactamase but is not easily influenced by IMP metallo- ⁇ -lactamase.
  • the at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase preferably comprise at least a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase and more preferably comprise at least: a ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase; a ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase; and a cephem ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase but is not easily influenced by VIM metallo- ⁇ -lactamase.
  • the antibacterial powers of the ⁇ -lactam agents decrease by the production of metallo- ⁇ -lactamase.
  • the degree of this decrease varies depending on the type of metallo- ⁇ -lactamase or the types of the ⁇ -lactam agents.
  • the degree of formation of an inhibition zone i.e., the shape of an inhibition zone derived from each ⁇ -lactam agent also differs depending on the type of metallo- ⁇ -lactamase.
  • the “ ⁇ -lactam agent that is easily influenced by metallo- ⁇ -lactamase” means a ⁇ -lactam agent that forms a smaller inhibition zone in the presence of each metallo- ⁇ -lactamase than that in the absence of metallo- ⁇ -lactamase.
  • ⁇ -lactam agent that is not easily influenced by metallo- ⁇ -lactamase means a ⁇ -lactam agent that forms an inhibition zone having a size unchanged or less changed even in the presence of each metallo- ⁇ -lactamase.
  • the level of this influence differs depending on the combination of metallo- ⁇ -lactamase and a ⁇ -lactam agent.
  • ⁇ -lactam agents can be classified according to the purpose.
  • the ⁇ -lactam agents can be classified and selected as follows with the level of this influence depending on these types of metallo- ⁇ -lactamases as selection criteria.
  • ⁇ -lactam agent examples include carbapenem ⁇ -lactam agents (e.g., biapenem and doripenem). This ⁇ -lactam agent is preferably biapenem.
  • Examples of the ⁇ -lactam agent that is easily influenced by both IMP metallo- ⁇ -lactamase and VIM metallo- ⁇ -lactamase include carbapenem ⁇ -lactam agents (e.g., imipenem and meropenem). This ⁇ -lactam agent is preferably imipenem.
  • cephem ⁇ -lactam agent examples include ceftazidime and cefepime. This ⁇ -lactam agent is preferably ceftazidime.
  • ⁇ -lactam agents include a ⁇ -lactam agent that is easily influenced by VIM metallo- ⁇ -lactamase but is not easily influenced by IMP metallo- ⁇ -lactamase.
  • Any ⁇ -lactam agent that is included in these classifications may be used, and the present invention is not limited to the ⁇ -lactam agents exemplified above.
  • the term “spotting” means that the drug of interest is disposed at the predetermined position.
  • the order of spotting is not particularly limited.
  • the metallo- ⁇ -lactamase inhibitor is first spotted, and next, the at least 3 or more ⁇ -lactam agents differing in sensitivity to metallo- ⁇ -lactamase can be spotted.
  • the metallo- ⁇ -lactamase inhibitor and each ⁇ -lactam agent can be spotted, for example, in a disk form.
  • the disk containing the metallo- ⁇ -lactamase inhibitor can be prepared using a filter paper of appropriate size and shape.
  • this disk can be prepared by impregnating a 1 ⁇ 4-inch round filter paper with the metallo- ⁇ -lactamase inhibitor, if necessary using a solvent.
  • Drug disks respectively containing the ⁇ -lactam agents are commercially available, and they can be used as the disks respectively containing the ⁇ -lactam agents. Even if corresponding commercially available products are absent, a filter paper of appropriate size and shape (e.g., a 1 ⁇ 4-inch round filter paper) impregnated with each of the ⁇ -lactam agents, if necessary using a solvent, can be used.
  • a filter paper of appropriate size and shape e.g., a 1 ⁇ 4-inch round filter paper
  • the amounts of the metallo- ⁇ -lactamase inhibitor and each ⁇ -lactam agent spotted can be determined appropriately with consideration given to the diffusibility or culture time (diffusion time) of each ⁇ -lactam agent on the solid medium surface and further to the strength of the inhibitory effect of the metallo- ⁇ -lactamase inhibitor on metallo- ⁇ -lactamase, etc.
  • the metallo- ⁇ -lactamase inhibitor represented by the formula (I) 100 ⁇ g of the ⁇ -lactam agent (A), 100 ⁇ g of the ⁇ -lactam agent (B), and 300 ⁇ g of the ⁇ -lactam agent (C) can be used to thereby form an inhibition zone appropriate for identification.
  • these amounts are given merely as a guideline and can be changed appropriately with consideration given to the shape, size, or the like of an inhibition zone formed around the ⁇ -lactam agent according to the type of the bacteria to be tested.
  • the positions at which the metallo- ⁇ -lactamase inhibitor and each ⁇ -lactam agent are respectively spotted can be set such that the distances from the disks respectively containing the ⁇ -lactam agents to the disk containing the metallo- ⁇ -lactamase inhibitor are equal on the same solid medium surface.
  • the “distance” means the distance from the center of the disk containing the metallo- ⁇ -lactamase inhibitor to that of each of the disks respectively containing the ⁇ -lactam agents.
  • these disks respectively containing the ⁇ -lactam agents can be disposed such that they are uniformly spaced on the circumference at an equal distance from the center.
  • the positions at which the metallo- ⁇ -lactamase inhibitor and each ⁇ -lactam agent are respectively spotted is preferably set such that the disk containing the metallo- ⁇ -lactamase inhibitor is disposed at the center of the solid medium surface and the disks respectively containing the ⁇ -lactam agents are respectively disposed at an equal distance from the disk containing the metallo- ⁇ -lactamase inhibitor.
  • the disks respectively containing the ⁇ -lactam agents can be disposed such that their centers are respectively positioned at an equal distance, for example, 20 mm, from the center of the disk containing the metallo- ⁇ -lactamase inhibitor and they are also uniformly spaced on the circumference at an equal distance from the center.
  • these ⁇ -lactam agents can also be disposed on separate solid media at an equal distance from the disk containing the metallo- ⁇ -lactamase inhibitor, without using the same solid medium.
  • the disks respectively containing the ⁇ -lactam agents and the disk containing the metallo- ⁇ -lactamase inhibitor are placed onto the surface of the solid medium coated with the bacteria to be tested, and then, the solid medium can be cultured.
  • Culture conditions that adhere to the standard method specified by the Japanese Society of Chemotherapy or the disk diffusion method specified by CLSI can be used.
  • the culture conditions can be determined appropriately with consideration given to the diffusion range of the ⁇ -lactam agents, etc., and can be set to within ranges of, for example, 35 to 37° C. and 12 to 36 hours.
  • each ⁇ -lactam agent and the metallo- ⁇ -lactamase inhibitor placed on the surface of the solid medium are diffused into the surface and inside of the solid medium.
  • an inhibition zone is formed around each ⁇ -lactam agent.
  • the degree of formation of an inhibition zone (i.e., the shape of an inhibition zone) derived from each ⁇ -lactam agent differs depending on whether or not the bacteria to be tested are metallo- ⁇ -lactamase-producing bacteria and the type of metallo- ⁇ -lactamase produced by the identified metallo- ⁇ -lactamase-producing bacteria.
  • the bacteria to be tested are MBL-producing bacteria, and, further, an IMP or VIM type or any other type of the identified MBL-producing bacteria can be determined based on the shape of the inhibition zone thus formed.
  • the shape of an inhibition zone formed around each ⁇ -lactam agent varies depending on the effect of the metallo- ⁇ -lactamase inhibitor (compound (I)).
  • the inhibition zone formed around each ⁇ -lactam agent can be divided into two subzones (i.e., an inhibition subzone formed on the side opposite to the compound (I)-containing disk and an inhibition subzone formed on the compound (I)-containing disk side) by measurement along the center lines of the ⁇ -lactam agent-containing and compound (I)-containing disks ( FIG. 1 ).
  • the inhibition subzone formed on the side opposite to the compound (I)-containing disk is not subject to the effect of the metallo- ⁇ -lactamase inhibitor.
  • its radius can be minimized in the whole inhibition zone (e.g., the numerical value obtained here can be defined as “A 1 ”, “B 1 ” and “C 1 ” for the ⁇ -lactam agents (A), (B) and (C), respectively (Table 1)).
  • the radius of the disk can be used.
  • the inhibition subzone formed on the compound (I)-containing disk side is subject to the effect of the metallo- ⁇ -lactamase inhibitor.
  • its radius can be maximized in the whole inhibition zone (e.g., the numerical value obtained here can be defined as “A 2 ”, “B 2 ” and “C 2 ” for the ⁇ -lactam agents (A), (B) and (C), respectively (Table 1)).
  • the radius of the disk can be used.
  • the distance from the center of the drug-containing disk to the compound (I)-containing disk can be used (e.g., “A 2 ” in the flow 8 in the flowchart of FIG. 2 represents the distance from the center of the ⁇ -lactam agent (A)-containing disk to the compound (I)-containing disk).
  • the degree of change in the shape of the inhibition zone caused by the effect of the metallo- ⁇ -lactamase inhibitor can be measured as the ratio between the minimum and maximum radiuses of the inhibition zone, i.e., the degree of change in radius. For example, it can be calculated by dividing the maximum radius of the inhibition zone by the minimum radius thereof (e.g., this calculated value can be defined as “A 3 ”, “B 3 ”, and “C 3 ”, respectively (Table 1)).
  • the “shape of the inhibition zone” can be determined by at least one value as an index selected from the minimum radius of the inhibition zone, the maximum radius of the inhibition zone, and the ratio between the minimum and maximum radiuses of the inhibition zone (degree of change in radius).
  • Whether or not the bacteria to be tested are metallo- ⁇ -lactamase-producing bacteria can be determined based on whether or not the presence of the metallo- ⁇ -lactamase inhibitor influences the size of the inhibition zone, i.e., the degree of change in radius.
  • the bacteria to be tested can be identified as metallo- ⁇ -lactamase-producing bacteria.
  • the bacteria to be tested are IMP metallo- ⁇ -lactamase-producing bacteria or VIM metallo- ⁇ -lactamase-producing bacteria can be determined based on the degree of change in radius in the inhibition zone of each ⁇ -lactam agent caused by the presence of the metallo- ⁇ -lactamase inhibitor.
  • the degree of change in radius in the inhibition zone of the ⁇ -lactam agent that is easily influenced by IMP metallo- ⁇ -lactamase becomes equal to or larger than the predetermined size due to the presence of the metallo- ⁇ -lactamase inhibitor, the bacteria to be tested can be identified as IMP metallo- ⁇ -lactamase-producing bacteria.
  • the bacteria to be tested can be identified as VIM metallo- ⁇ -lactamase-producing bacteria. Whether or not the bacteria to be tested are IMP metallo- ⁇ -lactamase-producing bacteria or VIM metallo- ⁇ -lactamase-producing bacteria can also be determined based on the range of the predetermined minimum or maximum numerical value.
  • a decision flow for determining whether or not the bacteria to be tested are metallo- ⁇ -lactamase-producing bacteria and, further, either an IMP or VIM type of the bacteria can be designed using the degree of change in radius and numerical values such as minimum and maximum radiuses (e.g., “A 1 ” to “A 3 ”, “B 1 ” to “B 3 ” and “C 1 ” to “C 3 ” in Table 1).
  • the specific numerical values used in identification differ depending on the solid medium used, the types of the metallo- ⁇ -lactamase and ⁇ -lactam agents, the radiuses of the disks, or the distance between the disks.
  • the decision flow can be designed individually according to a protocol for carrying out the method according to the present invention.
  • Metallo- ⁇ -lactamase-producing bacteria whose metallo- ⁇ -lactamase type has been confirmed can be used for the design of the decision flow.
  • a decision flow shown in FIG. 2 can be used.
  • bacteria that correspond to the flow 2 are determined to be MBL-producing bacteria; bacteria that correspond to the flows 3 to 5 can be identified as IMP MBL-producing bacteria; and bacteria that correspond to the flows 6 to 8 without corresponding to the flows 3 to 5 can be identified as VIM MBL-producing bacteria.
  • Bacteria that correspond to the flow 1 can possibly be identified by appropriately diluting a coating solution and conducting the same test again.
  • Bacteria that cannot be identified using the decision flow of FIG. 2 can be determined to be MBL-producing bacteria other than IMP and VIM types or non-MBL-producing bacteria including bacteria producing not MBL but serine- ⁇ -lactamase (flow 9 ).
  • kits for use in the identification of metallo- ⁇ -lactamase-producing bacteria for carrying out the method according to the present invention.
  • the method according to the present invention can be carried out conveniently.
  • the kit for use in the identification of metallo- ⁇ -lactamase-producing bacteria comprises at least a substrate in a plate form (strip form) on which ⁇ -lactam agents (A) to (C) are disposed (preferably, fixed) at an equal distance from a disk containing a compound (I) positioned at the center.
  • the substrate used in the kit according to the present invention can be a substrate in a thin plate form (strip form) on which ⁇ -lactam agents (A) to (C) can be disposed at an equal distance from a disk containing a compound (I) positioned at the center.
  • the substrate is preferably made of a water-unabsorbable material such as a plastic.
  • the substrate can be provided with calibration marks (e.g., at a 0.5-mm interval).
  • the disks used in the kit of the present invention can be the same disks as those described in the method of the present invention.
  • FIG. 3 One example of the kit according to the present invention is shown in FIG. 3 .
  • a substrate has three strips extended in different directions (structure with three branches) for respectively disposing disks at an equal distance from a disk containing a compound (I) positioned at the center.
  • the angle and size of the strips (branched structure) are not particularly limited and can be determined appropriately with consideration given to the size of the solid medium used, etc.
  • the concentration of the drug in each disk can be determined appropriately with consideration given to the same points as those described in the method of the present invention.
  • the drug names and sizes described in the diagram are described merely for illustration, and the kit of the present invention is not limited to them.
  • a method of identifying bacteria to be tested using the kit comprises placing the kit with its disk surface downward onto a surface of a solid medium coated with the bacteria to be tested, culturing the solid medium in the same way as in the method to form an inhibition zone, measuring the radius of the inhibition zone by visual observation using the calibration marks on the substrate, and identifying the bacteria to be tested as IMP or VIM MBL-producing bacteria or the other bacteria by using the decision flow.
  • This method can be performed in the same way as in the method of the present invention except that the kit is used.
  • a test shown below was conducted using, as bacteria to be tested, a total of 41 strains of Pseudomonas aeruginosa (including Pseudomonas putida ): IMP types (10 IMP-1-producing bacterial strains, 18 IMP-7-producing bacterial strains, 4 IMP-10-producing bacterial strains, and 2 IMP- ⁇ -producing bacterial strains) and VIM types (3 VIM-1-producing bacterial strains and 4 VIM-2-producing bacterial strains). These 41 strains were strains whose MBL type had already been identified by PCR or the like, and this Example was performed using them.
  • a Muller-Hinton agar medium was coated with a bacterial solution adjusted to McFarland standard turbidity of 0.5. No water droplet of the bacterial solution on the medium surface was confirmed, and round filter papers (diameter: approximately 6 mm) respectively containing 300 ⁇ g of a compound (I), 100 ⁇ g of biapenem as a drug (A), 100 ⁇ g of imipenem as a drug (B), and 300 ⁇ g of ceftazidime as a drug (C) were placed thereonto as drug-containing disks.
  • round filter papers (diameter: approximately 6 mm) respectively containing 300 ⁇ g of a compound (I), 100 ⁇ g of biapenem as a drug (A), 100 ⁇ g of imipenem as a drug (B), and 300 ⁇ g of ceftazidime as a drug (C) were placed thereonto as drug-containing disks.
  • the compound (I) used was a compound represented by the formula (I) wherein both R 1 and R 2 are an ethyl group, and M 1 is a sodium cation (disodium 2,3-diethylmaleate).
  • the compound (I) was placed at the center of the agar medium, while the drugs (A) to (C) were placed such that the center of each disk was 20 mm distant from the center of the compound (I). After overnight culture at 35° C., the minimum and maximum radiuses of a growth inhibition zone around each disk were measured, and the degree of change in radius was calculated according to Table 1.
  • the IMP or VIM MBL-producing bacteria were identified using a decision flow of FIG. 2 .
  • bacteria to be tested were IMP-1-producing Pseudomonas aeruginosa .
  • a Muller-Hinton agar medium was coated with the Pseudomonas aeruginosa , and disks were then placed thereonto.
  • the state after overnight culture at 35° C. is shown in FIG. 4 .
  • the upper disk contained biapenem (BIPM) (A); the lower right disk contained imipenem (IPM) (B); the lower left disk contained ceftazidime (CAZ) (C); and the central disk was a compound (I)-containing disk.
  • the compound (I) used was a compound represented by the formula (I) wherein both R 1 and R 2 are an ethyl group, and M 1 is a sodium cation (disodium 2,3-diethylmaleate). Black portions around the disks are inhibition zones. It is observed that the inhibition zone of each ⁇ -lactam agent is changed by the presence of the compound (I)-containing disk.
  • An inhibition zone radius positioned on the lateral side in relation to the center of each ⁇ -lactam agent-containing disk represents the minimum radius of the inhibition zone, and an inhibition zone radius closer to the center represents the maximum radius of the inhibition zone.
  • “A 1 ” is 5.5 mm
  • “B 1 ” is 6.5 mm
  • “C 1 ” is 3.5 mm
  • “A 2 ” is 16.0 mm
  • “B 2 ” is 14.0 mm
  • “C 2 ” is 14.5 mm. From these numerical values, “A 3 ”, “B 3 ”, and “C 3 ” are calculated to be 2.9, 2.2, and 4.1, respectively. When these numerical values are applied to the decision flow of FIG.
  • “A 3 ”, “B 3 ”, and “C 3 ” are not less than 1.2 and thus, do not correspond to the flow 1 in the decision flow. However, all of “A 3 ”, “B 3 ”, and “C 3 ” are 1.2 or more and thus correspond to the flow 2 in the decision flow. Thus, the bacteria to be tested are identified as MBL-producing bacteria. Furthermore, “C 1 ” which is 3 mm or more and “C 3 ” which is 3 or more, correspond to the flow 3 in the decision flow. Thus, the bacteria to be tested were identified as IMP MBL-producing bacteria.
  • An identification test was conducted using, as bacteria to be tested, a total of 9 strains of Pseudomonas aeruginosa (including Pseudomonas putida ): IMP types (3 IMP-1-producing bacterial strains, 2 IMP-7-producing bacterial strains, and 1 IMP- ⁇ -producing bacterial strain) and VIM types (1 VIM-1-producing bacterial strain and 2 VIM-2-producing bacterial strains).

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JP2000224998A (ja) 1999-02-04 2000-08-15 Kokuritsu Kansenshiyou Kenkyusho メタロ−β−ラクタマーゼ産生菌の判別方法
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